Use of recombinant polypeptides from the genus Lupinus as antimicrobial or insecticidal on plants

ABSTRACT

This invention is related to a protein from the seeds, cotyledons or plantlets of  Lupinus  genus, as well as to the way of producing the protein in recombinant form and of expressing the protein in genetically modified plants. The invention relates to methods of its use, or of any modification of the protein that maintains its biological properties, as a supplement in human or animal nutrition and as a fungicide, insecticide, growth promoter, fertilizer or in the preparation of genetically modified organisms.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Divisional of U.S. patent application Ser. No. 12/893,889,filed Sep. 29, 2010 (now U.S. Pat. No. 8,541,654), which is aContinuation-in-Part of U.S. patent application Ser. No. 11/996,279,filed Jan. 18, 2008 (now U.S. Pat. No. 8,106,252), which is a 371National Stage Application of International Application No.PCT/IB2006/052403, filed Jul. 13, 2006, claiming priority to PortuguesePatent Application Nos. 103511 and 103322, filed Jun. 28, 2006 and Jul.21, 2005. The entire disclosures of the prior applications areconsidered part of the disclosure and are hereby incorporated byreference.

FIELD OF THE INVENTION

This invention is included in the area of Biology, with its practicalapplicability as a fungicide, insecticide, growth promoter or fertilizerbelonging to the fields of Agronomical Sciences and Agriculture and asits applicability as a supplement in the diet of animals belonging tothe field of Human and Animal Nutrition.

BACKGROUND OF THE INVENTION

This invention relates to a polypeptide with antifungal, anti-Oomyceteand plant growth promoter properties, extracted from seeds, cotyledonsor plantlets of the genus Lupinus, and to its application in the controlof pathogenic agents that attack plants and as a plant bio-stimulant.This polypeptide may be applied directly on plants, or the plants may begenetically modified to express the polypeptide in their tissues. Inaddition, due to its unusual inherent characteristics, the polypeptidemay be utilized in the preparation of protein concentrates useful assupplements in the diet of man and other animals.

The present invention also describes the nucleotide DNA sequencecorresponding to the gene fragment that encodes the Lupinus polypeptide,as well as its sequence of amino acid residues, microorganismstransformed with the gene fragment that encodes the Lupinus polypeptideand methods for its application as a fungicide, insecticide, plantgrowth promoter or fertilizer or as a supplement in human or animalnutrition.

It is also an object of this invention the polypeptide characterized bythe sequence of amino acid residues referred above, in which one or moreamino acid residues are absent, have been substituted or added, ormaintaining its biological activities after suffering chemicalmodification, such as, for example, glycosylation.

The control of pathogenic agents constitutes a serious problem worldwidewith respect to the most important crops. Pathogenic fungi areparticularly important in what concerns the storage of agriculturalproducts. Presently, control over fungal growth is generally achieved bymassive applications of chemical fungicides. However,phytopharmaceutical products currently available in the market displayseveral serious disadvantages. On one hand, they exhibit high economicaland environmental costs; on the other hand, many fungal species havebeen developing resistance mechanisms to some top fungicides, oftenturning them obsolete in a couple of years after their introduction inthe market.

Even though plants do not possess an immunological system resemblingthat of animals, they have evolved an inherent resistance to the attackof pathogenic fungi. However, the techniques employed for plant growth,harvest and storage in modern agriculture promote very often good oroptimal conditions for pathogen development.

In addition, the number of microbial pathogens that may affect and harmplant crops is quite high. As an example, the following genera may bereferred: Alternaria, Ascochyta, Botrytis, Cercospora, Colletotrichum,Diplodia, Erysiphe, Fusarium, Gaeumanomyces, Macrophomina, Nectria,Phoma, Phomopsis, Phymatotrichum, Phytophthora, Plasmopara, Puccinia,Pythium, Rhizoctonia, Uncinula, and Verticillium. The application of thefungicides currently available in the market is limited to some of thesegenera, and is not an effective solution in the control of plantinfections.

An alternative strategy in the fight against microbial pathogens is theidentification and purification of substances of biological origin withpotent antifungal activity. The identification of such compoundsinvolves searching a variety of organisms, such as plants andmicroorganisms, for substances that are subsequently tested inantifungal assays and finally isolated and characterized.

In this way, many classes of antifungal proteins have already beenisolated, including chitinases, cystein-rich proteins that bind stronglyto chitin, β-1,3-glucanases, permeatins, thionins and lipid transferproteins. These proteins are thought to play a fundamental role in thenatural defenses of plants against the attack of pathogens.

Several methodologies are described in the available literature on theutilization of antifungal proteins, extracted from plants ormicroorganisms, either for direct application over the pathogenicagents, or in transgenic plants expressing those proteins. Theantifungal proteins most often utilized in these methodologies includechitinases, glucanases, osmotin-type proteins and lysozymes. Variousstudies have demonstrated that genetically modified plantsover-expressing these proteins exhibit enhanced resistance to manypathogens (EP 0392 225).

Modern techniques of Molecular Biology allowed the development ofrecombinant DNA technology and, consequently, plant transformation withgenes encoding antifungal proteins. This procedure usually involvesinsertion of the gene encoding the protein of interest in a planttissue, followed by regeneration of a whole plant from the geneticallymodified plant tissue.

However, the activity of some of these proteins is reduced by thepresence of ions, in particular potassium, sodium or calcium. For thisreason, although the proteins may exhibit a potent antifungal activityin in vitro assays, they may be ineffective in vivo due to the highphysiological concentrations of the ions that may naturally occur in thetransformed plant tissues.

Document “Ramos, Paula Cristina Rodrigues, et. al—“Accumulation of alectin-like breakdown product of beta-conglutin catabolism in cotyledonsof germinating Lupinus albus L. seeds” Planta (Heidelberg), vol. 203, n°1, 1997, pages 26-34″ describes isolation and purification of a 20-kDapolypeptide which is an intermediate breakdown product of beta-conglutincatabolism, the vicilin-like storage protein, which interacts with avariety of glycoproteins and possesses a lectin-type activity. In Table1 of this document, several N-terminal amino acid sequences aredescribed including the one of the 20-kDa polypeptide isolated from L.albus seeds. However, the N-terminal sequence presented in the Table 1of this document includes exclusively the identity of residues no. 5, 6,7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 20, and 23. This corresponds toa total of 15 residues out of the 173 amino acid residues that composeSEQ ID NO: 1 of the present invention. In fact, the N-terminal sequenceof this document is not the responsible for the biological propertiesrelated to the polypeptide of the present invention. Moreover, theN-terminal sequence described in the referred document is intended to beused to construct suitable primers, which may then be utilized inmolecular biology as the first step of the long and uncertain process ofgene and protein sequencing, while the main purpose of the presentinvention is to disclose a polypeptide exhibiting several biologicalactivities.

The document “Database UniProt [Online] 16 Aug. 2004 (2004 Aug.16)—“Beta-conglutin” discloses a protein obtained from Lupinus albus,namely beta-conglutin. This protein is the major seed storage proteinpresent in Lupinus seeds. Despite being the precursor of the polypeptidewith the sequence of SEQ ID NO:1, according to the present invention,these two polymers correspond to distinct molecules for a number ofreasons, namely a) beta-conglutin is regarded as a protein, with atypical quaternary structure, whereas the molecule with the sequence ofSEQ ID NO:1 should not be considered as a protein, but rather as apolypeptide; b) beta-conglutin, much like the vast majority of proteins,is easily destroyed by harsh conditions. On the opposite, thepolypeptide of the present invention is extremely resistant todenaturation, withstanding prolonged boiling, treatment with strongacids or bases, detergents and organic solvents. In addition, it isresistant to the Sun ultraviolet radiation; c) these two moleculesfulfil entirely different roles in nature—whereas beta-conglutin is aseed storage protein, the polypeptide of the present invention displaysa protective role in the plant, exhibiting a potent antifungal activityand a growth promoter activity. On the other hand, whereasbeta-conglutin possesses strong catalytic activities of chitosanase andchitinase, the polypeptide of the present invention exhibits catalyticactivity of beta-N-acetyl-D-glucosaminidase and a low level ofchitosanase activity.

Document “Database UniProt [Online] 24 May 2005 (2005 May24)—“Vicilin-like protein (fragment)” discloses a fragment (length: 531amino acid residues; molecular mass: 62,032 Da) of the same protein(i.e. beta-conglutin) described in the previous document (length: 533amino acid residues; molecular mass: 62,130 Da). Therefore, all remarksmade above in what concerns this document apply equally to the documentunder analysis.

In conclusion, it is becoming imperative the identification andpurification of novel compounds of biological origin exhibitinganti-pathogen properties in the fight against the pathogens that affectplants. Particular importance must be given to those compounds that areeffective over a wide range of pathogens and that maintain thebiological activity under in vivo conditions.

Agricultural practices have been optimized, over a long time period, topromote plant growth and development and to increase crop production. Onthe other hand, it is predictable that in the medium to long term, foodshortage may occur in many areas of the globe. Current techniques tocontrol plant growth under environmentally controlled conditions areexpensive and require complex equipment. For these reasons, manyresearchers have searched for and have reported physiologically activesubstances, either natural or synthetic, that exhibit a boosting effectupon the growth and development of crops. However, only a few of thesesubstances have found practical application under real, agriculturalconditions. Therefore, it is also increasingly important to discover orto develop plant growth promoters that are environmentally friend andthat present no toxicity to man, animals and the environment.

Plant legumes or, more specifically, their seeds, are considered as themajor source of protein worldwide for animal and human nutrition. Inthis respect, soybean fulfills a prominent role, not only for the highprotein content and quality of its seeds, but also for their richness inoil. However, from the agricultural point of view, soybean requiresfertile soils and an abundant water supply. Plants belonging to theLupinus genus have conquered, over the last few decades, a relevant,strong and of great potential position in comparison to soybean. If, onone hand, their seeds possess protein and oil levels comparable to thoseof soybean, on the other, their species are well adapted to poor soilsand to conditions of low water availability. For these reasons, lupinshave sometimes been considered as the “poor cousins” of soybean.

The high level of alkaloids which are toxic to animals and which arenaturally present in traditional, wild-type lupin seeds have longhampered the generalized cultivation of Lupinus species and the use oftheir seeds for animal and human consumption. This is the main reasonwhy lupin cultivation has lagged far behind that of soybean. InPortugal, for example, traditional consumption of lupin seeds has longbeen associated to beer ingestion. These seeds are first boiled in water(heating at 100° C. destroys the capacity of seeds to germinate but doesnot block the imbibition's process) and then immersed under runningwater for a few days to remove the toxic alkaloids. However, the recentapplication of breeding techniques allowed the development of the socalled sweet lupin varieties, characterized by containing a low seedalkaloid content (<0.004% w/w), as opposed to the traditional bittercultivars (alkaloid content >0.004% w/w). For this reason, the seeds ofsweet lupin varieties may be safely utilized as human and animal feed.

It is therefore predictable an increasing development of lupin-derivedfood products for both human and animal nutrition, as has happened anumber of decades ago with soybean. This is particularly important inthe case of lupin seed proteins, either albumins or globulins.

SUMMARY OF THE INVENTION

It is expected for the present invention to solve the technical problemassociated with the identification and purification of compounds ofbiological origin that are capable of controlling a wide range ofpathogenic agents that affect plant crops and that act as plant growthpromoters while maintaining their biological activities under in vivoconditions.

The solution is based on the characterization and identification of apolypeptide present in plants belonging to the genus Lupinus, thatexhibits the following unusual characteristics: (i) a potent antifungaland anti-Oomycete activity, which confers great potential as afungicide; (ii) a strong plant growth promoter activity, particularlynotorious on unhealthy or naturally weakened plants; (iii) an extremeresistance to denaturation, allowing its use under field conditions;(iv) a very high susceptibility to proteolysis, which makes it harmlessto the environment and nontoxic to man and animals; and (v) a wellbalanced amino acid composition.

Therefore, the first aspect of the present invention refers to thepolypeptide with the sequence: (5′ KIRVLER FDQRTNRLEN LQNYRIVEFQSKPNTLILPK HSDADYVLVV LNGRATITIV NPDRRQAYNL EYGDALRIPA GSTSYILNPDDNQKLRVVKL AIPINNPGYF YDFYPSSTKD QQSYFSGFSR NTLEATFNTR YEEIQRIILG NED3′) (SEQ ID NO: 3).

Another aspect of the invention refers to the polypeptide with thesequence of SEQ ID NO: 1

Another aspect of the present invention concerns a DNA fragment thatencodes that polypeptide. The invention also regards the use of thepolypeptide, or any preparation containing it, as a fungicide,insecticide, growth promoter or fertilizer, either by directapplication, in recombinant form or by expression in geneticallymodified organisms. Finally, the invention considers the use of thepolypeptide or any preparation containing it as a supplement in human oranimal nutrition.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1—Grapevine leaves heavily infected with powdery mildew weresprayed with water (leaf on the right) or with the native proteincontaining the polypeptide extracted from Lupinus (leaf on the left).(A) 24 h after spraying; (B) 2 months after spraying.

FIG. 2—Optical microscopy observations of germination of spores from thefungus responsible for powdery mildew in grapevine. Fungal spores werecarefully removed from the surface of young infected grapevine leavesand inoculated in water agar 0.6% (w/v). (A), (B) and (C)—Controls; (D)and (E)—addition of 200 μg of the total protein fraction from maturegrapes, containing pathogenesis-related (PR) proteins; (F) and(G)—addition of 200 μg of the native protein containing the polypeptideextracted from Lupinus. Each assay was observed after 24 and 48 h. Phasecontrast microscopy was employed and the magnification used was 125×.

FIG. 3—Metallurgic microscopic observations of grapevine leaves. (A)Healthy leaves; (B) leaves infected with powdery mildew; (C) leavesinfected with powdery mildew, 12 h after spraying with the nativeprotein containing the polypeptide extracted from Lupinus. Themagnification used is specified in each photograph.

FIG. 4—Effect of the polypeptide from Lupinus, produced in a recombinantform in Escherichia coli, on the germination and development of Uncinulanecator spores, the causal agent of powdery mildew.

FIG. 5—Rose plants in the same developmental stage were sprayed withwater (rose plant on the right) or with a solution containing thepolypeptide extracted from Lupinus (200 μg native protein containing thepolypeptide/mL; rose plant on the left). The photograph shows thedevelopmental stage of both plants three weeks after spraying.

FIG. 6—Watermelon plants produced in a nursery. Six weeks after theonset of germination the plants were sprayed with water (control; A), acrude Lupinus extract containing 100 μg native protein containing thepolypeptide/mL (B), a plant growth promoter commercially available inthe market (concentration recommended by the manufacturer) (C), and acrude Lupinus extract containing 200 μg native protein containing thepolypeptide/mL (D). The experiment was followed during two weeks and theplants photographed.

FIG. 7—Typical profile of the insolubility of the globulins from Lupinusgenus plants as a function of calcium and magnesium concentrations. Thisprofile is exemplified for the effect of these cations on theself-aggregation of the native protein containing the polypeptideextracted from Lupinus (▪) and of β-conglutin from Lupinus (∘).β-Conglutin (0.5 mg·mL⁻¹; ∘) and the native protein containing thepolypeptide extracted from Lupinus (0.5 mg·mL⁻¹; ▪) were purified fromdry seeds or from cotyledons detached from plantlets that weregerminated and grown for eight days, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a novel polypeptide with potentantifungal properties, which exhibits a powerful activity on thegermination and development of spores from fungal and Oomycete pathogensfor plants, and with plant growth promoter activity, particularlynotorious on unhealthy or naturally weakened plants. The invention alsoconsiders the use of the polypeptide or any preparation containing it asa supplement in human or animal nutrition. The DNA nucleotide sequenceof the gene fragment that encodes the polypeptide from Lupinus does notshare any significant homology with any other antifungalprotein/polypeptide that has been isolated from plants. The polypeptidefrom Lupinus constitutes a novel type of polypeptide among theantifungal proteins described in plants.

The polypeptide referred to in the present invention is purified fromcotyledons extracted from germinated seedlings of the genus Lupinus. Thepresent invention includes the description of the methodology employedto isolate the polypeptide from the plant tissues, the DNA nucleotidesequence of the gene fragment (SEQ ID NO: 2) that encodes it, and thecorresponding sequence of amino acid residues (SEQ ID NO: 1). Thecarboxy terminal region of the polypeptide is represented by SEQ ID NO:3.

(SEQ ID NO: 1)Arg Arg Gln Arg Asn Pro Tyr His Phe Ser Ser Gln Arg Phe Gln ThrLeu Tyr Lys Asn Arg Asn Gly Lys Ile Arg Val Leu Glu Arg Phe AspGln Arg Thr Asn Arg Leu Glu Asn Leu Gln Asn Tyr Arg Ile Val GluPhe Gln Ser Lys Pro Asn Thr Leu Ile Leu Pro Lys His Ser Asp AlaAsp Tyr Val Leu Val Val Leu Asn Gly Arg Ala Thr Ile Thr Ile ValAsn Pro Asp Arg Arg Gln Ala Tyr Asn Leu Glu Tyr Gly Asp Ala LeuArg Ile Pro Ala Gly Ser Thr Ser Tyr Ile Leu Asn Pro Asp Asp AsnGln Lys Leu Arg Val Val Lys Leu Ala Ile Pro Ile Asn Asn Pro GlyTyr Phe Tyr Asp Phe Tyr Pro Ser Ser Thr Lys Asp Gln Gln Ser TyrPhe Ser Gly Phe Ser Arg Asn Thr Leu Glu Ala Thr Phe Asn Thr ArgTyr Glu Glu Ile Gln Arg Ile Ile Leu Gly Asn Glu Asp (SEQ ID NO: 2)_(5′)CGTAGACAAAGGAACCCTTATCACTTCAGCTCTCAAAGATTCCAAACTCTTTACAAAAATAGGAATGGCAAAATCCGTGTGCTCGAGAGGTTTGACCAAAGAACCAATAGACTTGAGAATCTCCAAAACTACCGCATTGTTGAGTTCCAATCAAAACCTAACACTCTCATTCTCCCTAAACACTCTGATGCTGACTACGTCCTCGTTGTACTCAATGGTAGAGCCACAATCACGATAGTAAACCCTGATAGAAGACAAGCATATAACCTTGAGTATGGCGATGCTCTCAGAATCCCAGCTGGCTCAACTTCATATATCCTTAACCCGGATGACAACCAGAAGCTTAGAGTAGTCAAGCTCGCAATACCCATCAACAATCCTGGCTACTTTTATGATTTCTATCCATCGAGTACTAAAGACCAACAATCCTACTTCAGTGGCTTCAGCAGGAACACTTTAGAGGCCACCTTCAATACTCGTTATGAAGAGATACAAAGGATTATTTTAGGGAATGAGGAT3′ (SEQ ID NO: 3)_(5′)KIRVLER FDQRTNRLEN LQNYRIVEFQ SKPNTLILPK HSDADYVLVVLNGRATITIV NPDRRQAYNL EYGDALRIPA GSTSYILNPD DNQKLRVVKLAIPINNPGYF YDFYPSSTKD QQSYFSGFSR NTLEATFNTR YEEIQRIILG NED 3′

This polypeptide appears to occur naturally during only a very shortperiod in the lifetime of plantlets from Lupinus genus. It has beendemonstrated by the present inventors that β conglutin, the major seedstorage protein from Lupinus genus, is the biosynthetic precursor of thepolypeptide from Lupinus. Indeed, the Lupinus polypeptide is a highlyprocessed polymer that has undergone several levels of chemicalmodification. This increased tremendously the difficulty of its study,including the sequencing of amino acid residues and of the correspondingnucleotides. During seed formation, the gene encoding β conglutin istranscribed into the corresponding mRNA, whose translation results inthe synthesis of the biosynthetic precursor of β conglutin. Thisprecursor is then extensively processed, including glycosylation, fromwhich the several tens of different types of subunits that compose βconglutin are produced. In the following cycle of vegetative growth,several days after the onset of germination, the initial steps in thecatabolism of β conglutin involve proteolytic cleavage of all or most ofits constituent subunits, resulting in the accumulation of a nativeprotein, as the major subunit, corresponding to the polypeptidedescribed in this invention. Due to its intrinsic antifungal properties,which are naturally exploited by the host plant, this polypeptide ismaintained in very high concentrations in the cotyledons of thedeveloping plants, during a life stage in which the plant is mostsensitive to fungal and insect attack. After a few days, the polypeptideis degraded and its amino acids used in the growth of the young plant.

The Lupinus polypeptide described in the present invention exhibits someproperties that distinguish it from the other antifungal proteinsdescribed in the literature. This makes it a promising target with greatpotential to develop an efficient method to control the fungi thataffect plants and/or animals:

(1) Potent antifungal and anti-Oomycete activity, which confers greatpotential to the polypeptide as a fungicide,

(2) Strong plant growth promoter activity, particularly notorious onunhealthy or naturally weakened plants,

(3) Extreme resistance to denaturation, which allows the use of thepolypeptide under field conditions,

(4) Great susceptibility to proteolytic attack, which makes it harmlessto the environment and nontoxic for man, and

(5) A well-balanced amino acid composition.

The polypeptide may also be utilized as an insecticide, growth promoteror fertilizer, and as a food supplement in human or animal nutrition.

In one embodiment, the polypeptide of the invention has the amino acidsequence of SEQ ID NO:1 wherein one or more amino acid residues aredeleted, substituted, added or modified, and the polypeptide maintainsantifungal, anti-Oomycete, insecticidal or plant growth promotingactivity. In one embodiment, the polypeptide is represented by asequence having not more than 50 deleted, substituted, added or modifiedamino acid residues to SEQ ID NO: 1, such as not more than 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, or 49 amino acid residues are deleted,substituted, added or modified.

In one embodiment, the polynucleotide of the invention has a modifiedsequence in which one or more nucleotides are deleted, substituted,added or modified, and which encodes a polypeptide with antifungalactivity, anti-Oomycete activity, insecticidal or plant growth promoteractivity. In one embodiment, the polynucleotide is represented by asequence having not more than 50 deleted, substituted, added or modifiednucleotides to SEQ ID NO: 2, such as not more than 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, or 49 nucleotides are deleted, substituted, added ormodified.

Sequence identity: The similarity between amino acid sequences orbetween nucleic acid sequences can be expressed in terms of thepercentage of conservation between the sequences, referred to assequence identity. Sequence identity is frequently measured in terms ofpercentage identity (or similarity or homology); the higher thepercentage, the more similar the two sequences are. In one embodiment,homologues or variants encompassed by the invention will possess arelatively high degree of sequence identity when aligned to SEQ ID NOs:1 or 2 using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet., 6:119, 1994, presents adetailed consideration of sequence alignment methods and sequenceidentity calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBL Bethesda, Md.)and on the NCBI website on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.A description of how to determine sequence identity using this programis available on the NCBI website. Other specific, non-limiting examplesof sequence alignment programs specifically designed to identifyconserved regions of genomic DNA of greater than or equal to 100nucleotides are PIPMaker (Schwartz et al., Genome Research 10:577-586,2000) and DOTTER (Erik et al., Gene 167:GC1-10, 1995).

In one embodiment, the polypeptide of the invention has a sequence of atleast 75% sequence identity to the sequence set forth in SEQ ID NO: 1,such as, but not limited to at least 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 99.9% sequence identity to the polypeptide set forthin SEQ ID NO: 1.

In one embodiment, the polynucleotide of the invention has a sequence atof least 75% identity to the sequence set forth in SEQ ID NO: 2, suchas, but not limited to at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 99.9% sequence identity to the polynucleotide set forth inSEQ ID NO: 2.

In one embodiment, homologues and variants of SEQ ID NOs: 1 and 2encompassed by the invention are typically characterized as having apercentage sequence identity counted over the full length alignment withthe corresponding sequence of SEQ ID NOs: 1 or 2 using, in oneembodiment, the NCBI Blast 2.2.23, set to default parameters. Methodsfor determining sequence identity over short windows are available atthe NCBI website on the Internet. One of skill in the art willappreciate that these sequence identity ranges are provided for guidanceonly; it is entirely possible that strongly significant homologues couldbe obtained that fall outside of the ranges provided.

In another embodiment, the peptide of the invention has not more than 50conservative substitutions to SEQ ID NO: 1, such as not more than 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, or 49 conservative substitution arepresent.

Two practical problems in present-day agriculture are the growthreduction or inhibition observed in unhealthy or naturally weakenedplants and the toxicity normally associated to available bio-stimulants.The polypeptide extracted from Lupinus plant tissues exhibits a stronggrowth promoter activity upon plant growth and development. Indeed,Lupinus preparations or extracts containing the polypeptide possess astrong bio-stimulatory activity upon all tested plants, including, forexample, grapevine, rose, watermelon and tomato. This effect isnotorious for concentrations of the native protein containing thepolypeptide equal to or above 200 μg/mL. The other components present innon-pure extracts of the Lupinus polypeptide add value to thepreparations because they act as a foliar fertilizer. The absence oftoxicity of the Lupinus polypeptide for man, animals and the environmentindicate that its applications in agriculture do not have any damagingeffect on the environment.

Another aspect of the present invention concerns the methodologyutilized for the recombinant production of the Lupinus polypeptide inbacteria, with the aim of expressing it constitutively in geneticallymodified plants. Eventually, these plants will present a high level ofresistance to pathogenic fungi, namely with respect to the fungi ofdifficult control (as is the case of the fungi responsible for the socalled wood diseases), against which the traditional fungicides ofexogenous application are not at all effective.

The polypeptide was extracted from eight-days-old Lupinus albus cv.LeBlanc pantlets. The seeds were placed in a constant temperature room(25° C. day, 20° C. night) with a photoperiod of 16 h light/8 h dark.

After harvest, the cotyledons were frozen in liquid nitrogen.polypeptide extraction was performed in 100 mM Tris-HCl buffer, pH 7.5,containing 10% (w/v) NaCl, 10 mM EDTA (ethylenediamine-tetraacetic acid)and 10 mM EGTA (ethylene glycol bis((3-aminoethylether)-N,N,N′,N′-tetraacetic acid). After an incubation period of 30minutes at 4° C., the extract was centrifuged at 30,000 g, for 1 h at 4°C. The supernatant was desalted and the native protein containing thepolypeptide extracted from Lupinus subsequently purified by FPLC (FastProtein and Peptide Liquid Chromatography)-anion exchangechromatography.

N-terminal sequencing of the polypeptide extracted from Lupinus wasaccomplished by Edman degradation. The obtained sequence of amino acidresidues was used to design degenerate primers. Total mRNA was extractedfrom developing Lupinus albus seeds at a stage where maximal synthesisof the β conglutin precursor occurs. The mRNA extraction was performedusing protocols/kits for mRNA purification from plant material. The cDNAcorresponding to the gene fragment encoding the polypeptide extractedfrom Lupinus was amplified by PCR (polymerase chain reaction) using thedegenerate primers previously designed. Using the nucleotide sequenceobtained as a template, new primers were designed and the completenucleotide sequence of the gene fragment encoding the polypeptideextracted from Lupinus was achieved by the 3′ and 5′ RACE (rapidamplification of cDNA ends) technique.

The polypeptide from Lupinus was produced in recombinant form in thebacterium Escherichia coli. The gene fragment encoding the polypeptidefrom Lupinus was cloned in a suitable vector, allowing the associationof the gene of interest to the promoter T7lac. This promoter isinductive; therefore, the expression of the genes that are associatedwith it occurs exclusively in the presence of the sugarisopropylthio-β-galactoside. Finally, competent Escherichia coli cellswere transformed.

As described above, the polypeptide from Lupinus was obtained in arecombinant form from bacteria. However, to be tested for its antifungalactivity, the recombinant polypeptide from Lupinus had to be isolatedfrom all other bacterial proteins/polypeptide. To this end, thepolypeptide from Lupinus was previously produced in a recombinant formwith a tag of histidine residues (His-Tag). The methodology employed forits purification was based on the high affinity of the nickel ions forthe His-Tag. In this way, having nickel ions bound to an agarose matrix,the purification of the recombinant polypeptide was accomplished knowingthat among all the proteins/polypeptide present in the bacterial totalextract, only the polypeptide from Lupinus binds to the agarose matrix.Subsequently, the polypeptide from Lupinus was recovered after asuitable set of washes and elutions, and the His-Tag removed aftertreatment with an appropriate proteolytic enzyme.

The careful choice of a suitable promoter is a pre-requisite for plantgenetic modification. Several types of promoters are described in theliterature, allowing the expression of the associated genes. To expressthe gene fragment encoding the polypeptide described in the presentinvention, the selected promoter may be inductive or constitutive,depending on the type of expression required. The choice of the promoteris also important to direct the synthesized polypeptide to the tissue orcellular compartment selected (post-transcriptional modifications).

Plant transformation may be achieved by different methodologies, suchas, for example, plant transformation via Agrobacterium, protoplasttransformation, gene transfer to pollen grains, direct injection intoreproductive organs or immature embryos, and particle bombardment. Eachof these methods presents specific advantages and disadvantages.Nevertheless, they all have already been utilized in different planttypes.

To transform plants with the gene fragment encoding the polypeptide fromLupinus, the selected method was transformation via Agrobacterium(Fraley et al., 1983), using a suitable expression vector, containing acodifying region for the gene of interest associated to an appropriatemarker gene. Plant regeneration, plant development and plant transfer tothe culture medium from a single protoplast may be achieved followingseveral methodologies available in the literature. This process includesseveral steps on the selection of transformed cells and the subsequentculture of these cells by the usual methods employed in the developmentof embryogenic cultures. The regenerated plantlets are finally grown ina suitable culture medium, usually soil.

It is also object of the present invention any agricultural formulationthat includes as the active ingredient the polypeptide with the sequenceof SEQ ID NO: 1, glycosylated, phosphorylated, alkylated and/orprenylated, or a recombinant form of the polypeptide obtained bytransformation of cells characterized by being utilized in prevention,control and fight of pathogenic fungi or Oomycetes or plagues caused byinsects, or as a growth promoter or fertilizer.

Another aspect of the present invention is related to the frequentreduced levels in plant proteins/polypeptides of the human and animaldiets and, in some cases, to the poor protein digestibility and animbalanced amino acid composition. In fact, crude preparationscontaining the Lupinus polypeptide possess not only a major globulin(the Lupinus native protein containing the polypeptide, object of thepresent invention) but also a variety of albumins that are naturallypresent in the plant material utilized in the protein extraction.Therefore, these crude preparations of the Lupinus polypeptide areparticularly rich in proteins and may be utilized as a proteinsupplement in animal or human nutrition as tofu (after globulinprecipitation with calcium and magnesium) or as ricotta (after heatprecipitation of the albumins).

The analysis of the amino acid composition of the Lupinus polypeptideand its great susceptibility to all tested proteases (including trypsin,chymotrypsin, subtilisin, proteinase K and pronase) indicate that thispolypeptide has a high nutritive value for animals. However, thepolypeptide considered in the present invention is a globulin. For thisreason, the Lupinus polypeptide or the native protein containing it areinsoluble in water and in dilute salt solutions, but readily soluble inhigh ionic strength solutions. Nevertheless, legume globulins areinsoluble only when in the presence of calcium, magnesium and otheralkaline-earth cations (Ferreira et al., 1999). These divalent cations,positively charged at neutral pH values, act as electrostatic bridgesbetween negatively charged globulin molecules, promoting or inducingtheir self-aggregation into complexes that are so large that areinsoluble (Ferreira et al., 1999; Ferreira et al., 2003). Tofu, forexample, is a curd similar to cheese or cottage cheese prepared byadding calcium and magnesium ions to a heated extract of soya beans.Both cations are routinely utilized in tofu preparation and arecommercially available in the form of Nigari®. In this way, a crudepreparation of the Lupinus polypeptide, containing both globulins andalbumins, may be used in the preparation of Lupinus globulinconcentrates after their precipitation with calcium and/or magnesium.FIG. 7, for example, shows the precipitation pattern of the Lupinusnative protein containing the polypeptide (▪) as a function of addedcalcium and magnesium concentrations. For comparative purposes, it isalso presented the precipitation profile of its precursor protein,β-conglutin (the main storage protein present in Lupinus seeds; ∘). Thealbumins that remain in the resulting serum may also be recovered, forexample, by heat precipitation, in a process similar to that utilized inthe preparation of ricotta (heat precipitation of the milk albumins thatremain in the serum after casein removal during cheese making). In thisway, preparations containing the Lupinus polypeptide may be utilized asa protein supplement in the human or animal diet.

To understand the potential of the present invention, several practicalexamples follow. However, these examples are not limiting in the sensethat alternative methodologies may be employed in the utilization of thepolypeptide from Lupinus as an agent of antifungal and Oomycete control,as an insecticide, as a growth promoter, as a fertilizer or as a proteinsupplement in the human or animal diet.

PRACTICAL EXAMPLES Examples 1 and 2

Effect of spraying the native protein containing the polypeptide fromLupinus on the surface of grapevine leaves infected with the fungusUncinula necator (the causal agent of powdery mildew in grapevine).

The antifungal activity of the polypeptide from Lupinus was evaluatedafter spraying the surface of a grapevine leaf with a solutioncontaining 200 μg of pure native protein containing the polypeptide/mL.As a control, a similar leaf was sprayed under identical conditions withwater. The results obtained are presented in FIG. 1 and show that thegrapevine leaf remains healthy two months after spraying the leaves withthe native protein containing the polypeptide, without traces of thefungus presence, even though the sprayed leaves were always andpermanently kept in close contact with heavily infected leaves.

Another trial was performed following an identical methodology, but theobservations of the surfaces of the treated grapevine leaves were madeusing a metallurgic microscope (FIG. 3).

Example 3

Effect of the native protein containing the polypeptide from Lupinus onthe germination and development of spores from Uncinula necator.

Spores from the fungus Uncinula necator were removed from the surface ofinfected grapevine leaves and inoculated in water agar 0.6% (w/v),containing 200 μg of pure native protein containing the polypeptide fromLupinus per mL, or 200 μg of the total protein fraction from maturegrapes (containing PR proteins) per mL. Spore germination anddevelopment of the germ tubes were followed by optical microscopy usingthe contrast phase lens system, during 24 and 48 h. The resultsobtained, presented in FIG. 2, show that a marked reduction occurred inthe presence of the medium containing the polypeptide from Lupinus, notonly in the number of germinated spores, but also in the length of thegerm tubes. This effect is notorious when compared with the resultobserved in the presence of the PR proteins.

Example 4

Effect of the native protein containing the polypeptide from Lupinus onthe germination and development of spores from the fungus Phomopsisviticola (the causal agent of excoriosis in grapevine).

Spores from the fungus Phomopsis viticola were inoculated in PDA (potatodextrose agar) medium. After 15 minutes, the spores were removed andmixed with a solution containing the native protein containing thepolypeptide from Lupinus in a final volume of 25 μL. These droplets wereplaced in Petri dishes and overlaid with glass slides which weresubsequently sealed, creating a wet chamber. Spore development wasfollowed by optical microscopy observations. A clear inhibition of sporegermination was evident. After 24 h developing hyphae suffered lysis.

Example 5

Effect of the recombinant polypeptide from Lupinus on the germination ofspores from the fungus Uncinula necator.

The recombinant polypeptide from Lupinus, expressed in bacteria, waspurified and its antifungal activity tested. These assays were performedas previously described in examples 2 and 3. The results obtained,presented in FIG. 4, show that the recombinant polypeptide exhibitidentical antifungal properties to those observed for the polypeptideextracted from Lupinus plants. After a 48 h incubation period in thepresence of the recombinant polypeptide from Lupinus, destruction of thespore cell walls is observed.

Example 6

Effect of the recombinant polypeptide from Lupinus on the germination ofspores from the Oomycete Plasmopara viticola (the causal agent of downymildew).

Spores from the Oomycete Plasmopara viticola were removed from thesurface of grapevine infected leaves and placed in water agar 0.6%(w/v), containing 200 μg of recombinant pure polypeptide from Lupinusper mL. Spore germination was followed during 48 h by optical microscopyobservations. Spore germination in water agar was used as a control.After 24 h, the spore cell walls were destroyed, with the concomitantrelease of the cellular content.

Example 7

Effect of spraying the native protein containing the polypeptideextracted from Lupinus on rose plants.

The bio-stimulant activity of the Lupinus native protein containing thepolypeptide was evaluated after spraying the leaf surfaces of a roseplant with a crude Lupinus extract containing 200 μg native proteincontaining the polypeptide/mL. As a control a rose plant in an identicaldevelopmental stage and incubated under the same environmentalconditions was sprayed with water. The result obtained, photographedthree weeks after spraying, is presented in FIG. 5 and shows a superiorgrowth for the plant sprayed with the Lupinus polypeptide, evidencing apremature appearance of the first floral buds.

Example 8

Effect of spraying the native protein containing the polypeptideextracted from Lupinus on nursery watermelon plants.

The bio-stimulant activity of the Lupinus polypeptide was evaluatedafter spraying the leaf surfaces of six-weeks-old nursery watermelonplants with a crude Lupinus extract containing 200 μg native proteincontaining the polypeptide/mL. The assay was performed under greenhouseconditions and the plants were sprayed with water (control; A); a crudeLupinus extract containing 100 μg native protein containing thepolypeptide/mL (B); a plant growth promoter commercially available inthe market (concentration recommended by the manufacturer) (C); and acrude Lupinus extract containing 200 μg native protein containing thepolypeptide/mL (D). Twenty-four plants were used in each assay. Theassay was followed during the subsequent two weeks and the resultsobtained are presented in FIG. 6. The plants sprayed with the highestconcentration of the Lupinus polypeptide (200 μg native proteincontaining the polypeptide/mL; D) exhibit the greatest development and asuperior leaf growth when compared with plants treated with water orwith the plant growth promoter commercially available in the market. Theplants sprayed with the lowest concentration of the Lupinus polypeptide(100 μg native protein containing the polypeptide/mL; B) exhibit a lowerlevel of development but still higher than that observed for the plantssprayed with water alone. The recommended level of application istherefore spraying the plants with a crude preparation of the Lupinuspolypeptide containing 200 μg native protein containing thepolypeptide/mL.

Example 9

Effect of spraying the native protein containing the polypeptideextracted from Lupinus on grapevine plants infected with Uncinulanecator (the fungal causal agent of powdery mildew, economically themost important disease of grapevine worldwide).

A crude Lupinus extract was prepared containing 200 μg of the Lupinusnative protein containing the polypeptide per mL. Grapevine plantsinfected with Uncinula necator and kept under greenhouse conditions weresprayed with the extract or with water (control). Twenty-four hoursafter application, the sprayed plants were observed—relative to thecontrol, the plants sprayed with the Lupinus polypeptide exhibited ahigher vigor and revealed the appearance of new shoots. This situationwas maintained during at least one week, after which the plants,previously weakened by the presence of the fungus, were exuberant andwith many new leaves with no symptom at all of the disease.

Example 10

Determination of the optimal calcium and magnesium concentrationsrequired for the preparation of a tofu-type Lupinus native proteincontaining the polypeptide concentrate.

The well-balanced amino acid composition of the Lupinus native proteincontaining the polypeptide, as well as its excellent digestibility (thepolypeptide is readily hydrolyzed into its component amino acids by theaction of the human digestive tract proteases) highlight the greatnutritive potential of the Lupinus polypeptide concentrate preparedafter precipitation with 5 mM calcium+magnesium of the globulins presentin a crude preparation of the Lupinus native protein containing thepolypeptide (see FIG. 7).

REFERENCES

-   Fraley, R. T., Rogers, S. G., Horsch, R. B., Sanders, P. R.,    Flick, J. S., Adams, S. P., Bittner, M. L., Brand, L. A., Fink, C.    L., Fry, J. S., Galluppi, G. R., Goldberg, S. B., Hoffman, N. L. &    Woo, S. C. (1983). Expression of bacterial genes in plant cells.    Proceedings of the National Academy of Sciences USA, 80, 4801-4807.-   Ferreira, R. B., Franco, E. & Teixeira, A. R. (1999). Calcium- and    magnesium-dependent aggregation of legume seed storage proteins.    Journal of Agricultural and Food Chemistry, 47, 3009-3015.-   Ferreira, R. B., Freitas, R. L. & Teixeira, A. R. (2003).    Self-aggregation of legume seed storage proteins inside the protein    storage vacuoles is electrostatic in nature, rather than    lectin-mediated. FEBS Letters, 534, 106-110.

What is claimed is:
 1. A transformed cell comprising a recombinantvector, wherein said vector comprises a DNA fragment comprising anucleotide sequence encoding a polypeptide, the polypeptide comprisingthe amino acid sequence of SEQ ID NO:1 or a sequence having at least 85%sequence identity thereto, wherein the polypeptide does not comprise asequence of 531 consecutive amino acid residues of β conglutin fromLupinus albus, and wherein the polypeptide exhibits at least one of thegroup consisting of: anti-fungal activity, anti-Oomycetes activity,insecticide activity, plant growth promoter activity, and nutritionalproperties.
 2. The transformed cell of claim 1, wherein the cell isderived from Escherichia coli.
 3. The transformed cell of claim 1,wherein the cell is derived from a plant or microorganism.
 4. Atransgenic plant comprising the transformed cell of claim 1, wherein thetransgenic plant exhibits resistance to pathogenic fungi, Oomycetes orplagues caused by insects, being obtained by expression of thepolypeptide in the transformed cell.
 5. A method to produce apolypeptide, the polypeptide comprising the amino acid sequence of SEQID NO: 1 or a sequence having at least 85% sequence identity thereto,wherein the polypeptide does not comprise a sequence of 531 consecutiveamino acid residues of β conglutin from Lupinus albus, and wherein thepolypeptide exhibits at least one of the group consisting of:anti-fungal activity, anti-Oomycetes activity, insecticide activity,plant growth promoter activity, and nutritional properties, the methodcomprising incubating a culture of the transformed cell of claim 1, andrecovering the polypeptide from a cell culture or a cell extract.